The present invention relates to animal models useful for the study of lipid metabolism that have been genetically modified to express or mis-express proteins involved in the sterol regulatory element binding protein (SREBP) pathway. The invention also relates to novel SREBP pathway nucleic acid and polypeptide sequences and their uses.
Triglycerides, phospholipids, and cholesterol, which form the three major classes of lipid, perform a variety of necessary functions in cell metabolism and arc vital constituents of biological membranes. However, elevated levels of lipids and/or improper lipid metabolism have been implicated in a variety of health disorders. Of particular concern mis increased blood cholesterol which leads to atherosclerosis (the deposition of cholesterol on arterial walls). This in turn may lead to heart disease, stroke or other disorders of the circulatory system. Accordingly, there is much interest within the pharmaceutical industry to understand the mechanisms involved in cholesterol synthesis and metabolism, particularly on the molecular level, so that blood cholesterol lowering drugs can be developed for the treatment or prevention of atherosclerosis.
Recent advances have been made in understanding some of the mechanisms involved in mammalian lipid metabolism. A key component is the sterol regulatory element binding protein (SREBP) pathway. SREBPs are transcription factors that activate genes involved in cholesterol and fatty acid metabolism. In the cholesterol biosynthetic pathway of vertebrates, SREBPs directly activate transcription of the genes encoding 3-hydroxy-3-methylglutaryl (HMG) coenzyme A synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. In the fatty acid and triglyceride biosynthetic pathways, the direct targets of SREBPs include fatty acid synthase, acetyl-CoA carboxylase, glycerol-3-phosphate acyltransferase, and acyl-CoA binding protein. Additionally, SREBPs modulate transcription of stearoyl CoA desaturase-1 and lipoprotein lipase. SREBPs also directly activate transcription of the gene encoding the low density lipoprotein (LDL) receptor, which provides cholesterol and fatty acids through receptor-mediated endocytosis. SREBPs are also implicated in the process of fat cell differentiation and adipose cell gene expression, particularly as transcription factors that can promote adipogenesis in a dominant fashion (reviewed by Spiegelman et al., Cell (1996) 87:377-389).
In high sterol conditions, SREBPs are retained as membrane-bound protein precursors that are kept inactive by virtue of being attached to the nuclear envelope and endoplasmic reticulum (ER) and therefore, excluded from the nucleus. As depicted in FIG. 1A, an SREBP in its membrane-bound form has large N-terminal and C-terminal segments facing the cytoplasm and a short loop projecting into the lumen of the organelle. The N-terminal domain is a transcription factor of the basic-helix-oop-helix-eucine zipper (bHLH-Zip) family, and contains an xe2x80x9cacid blobxe2x80x9d typical of many transcriptional activators. (Brown and Goldstein, Cell (1997) 89:331-340)
The N-terminal acid blob is followed by a basic helix-loop-helix-leucine zipper domain (bHLH-Zip) similar to those found in many other DNA-binding transcriptional regulators. bHLH-Zip domains have two functions: the helix-loop-helix subdomain mediates dimerization, and the basic region binds to specific DNA sequences tat include a direct repeat of 5xe2x80x2-PyCAPy-3xe2x80x2. SREBP binds to the sequence 5xe2x80x2-ATCACCCCAC-3xe2x80x2 (SEQ ID NO:30) which is known as xe2x80x9csterol regulatory element 1xe2x80x9d (SRE-1) and is upstream of the LDL receptor gene.
SREBPs are unique among bHLH-Zip proteins by virtue of the C-terminal domains attached to the bHLH-Zip domain. These include (fromxe2x80x94to C-terminus): (1) a hydrophobic membrane-spanning sequence of about 20 amino acids, (2) a hydrophilic stretch of about 31 amino acids that projects into the lumen of the ER, (3) a second hydrophobic membrane-spanning domain of about 20 amino acids, and (4) a C-terminal domain which, in vertebrates, has been determined to be required for sterol regulation of SREBP cleavage.
In low sterol conditions, the acid blob/bHLH-Zip domain of SREBP is released from the membrane after which it is rapidly franslocated into the nucleus and binds specific DNA sequences to activate transcription. Two sequential proteolytic cleavages are involved. Referring to FIG. 1B, a first protease, referred to as the site 1 protease (S1P) cleaves SREBP at approximately the middle of the lumenal loop. S1P has been cloned from Chinese hamster ovary (CHO) cells (GI (GenBank Identifier No. (hereinafter xe2x80x9cGIxe2x80x9d) 3892203) and a human cell line (GI4506774) (Sakai et al., J. Biol. Chem (1998) 273:5785-5793), and encodes a membrane bound glycoprotein of 1052 amino acids with subtilisin-like sequence features.
After cleavage at site 1, a second protease (the site 2 protease, S2P) cleaves the N-terminal fragment and releases the mature N-terminal domain into the cytosol, from which it rapidly enters the nucleus, apparently with a portion of the transmembrane domain still attached at the C-terminus. Mature, transcriptionally active SREBP is rapidly degraded in a proteosome-dependent process. This combination of proteolytic processing and rapid turnover allows the SREBP system to rapidly respond to changes in cellular membrane components. S2P homologues have been identified in both vertebrates and invertebrates and have been cloned from human cells and hamster cells (Rawson et al., Molec Cell (1997) 1:47-57). It is a membrane protein containing an HEXXH sequence characteristic of zinc metalloproteases. This family of proteins has high hydrophobicity throughout the amino acid sequence, suggesting the existence of several membrane-spanning regions.
A third component of the processing system for SREBPs is called SREBP Cleavage Activating Protein (SCAP). SCAP is a large transmembrane protein that activates S1P in low-sterol conditions. The N-terminal 730 amino acids have alternating hydrophobic and hydrophilic sequences which are predicted to form up to eight membrane spanning sequences separated by short hydrophilic stretches. This domain is strikingly similar to a domain of HMG CoA reductase (Hua et al., Cell (1996) 87:415-426) which is necessary to impart sterol regulation. In low sterol conditions, HMG-CoA reductase is quite stable, but when sterols are added the enzyme is rapidly degraded. It is believed that the membrane-spanning domain in SCAP, like its counterpart in HMG CoA reductase, can sense the levels of sterol in the ER membrane, either directly or indirectly.
The C-terminal domain of SCAP is hydrophilic and is made up of about 550 amino acids organized into four WD repeats. Recent work has demonstrated that these WD repeats bind directly to the C-terminal regulatory domain of SREBP suggesting that SCAP and SREBP are part of a stable complex in the membrane of the ER (Sakai et al., supra). It is likely that S1P and perhaps S2P are also part of the complex since SCAP is essential for activation of S1P activity. This SREBP processing complex is depicted in FIG. 2.
The involvement of the SREBP pathway in the regulation of cholesterol metabolism is of interest not only because excess blood cholesterol can lead to atherosclerosis, but also because there seem to be parallels between the processing of SREBPs and the processing of xcex2-amyloid precursor protein which has been implicated in Alzheimer""s disease (Brown and Goldstein, supra). To date, the SREBP pathway has been studied primarily using mammalian cell culture, by the isolation of mutant cells that are defective in regulation of cholesterol metabolism or intracellular cholesterol trafficking. The mutants can then serve as hosts for cloning genes by functional complementation. This has led to the molecular cloning of the S1P, S2P and SCAP genes (Rawson et al., supra; and et al., supra; and Goldstein et al., U.S. Pat. Nos. 5,527,690 and 5,891,631).
Some SREBP pathway genes have been identified in invertebrates. The isolation of a Drosophila SREBP, referred to as xe2x80x9cHLH 106xe2x80x9d (GI079656) has been described (Theopold et al., Proc. Natl. Acad. Sci., USA, (1996) 93(3):1195-1199). An expressed sequence tag (EST) from C. elegans which has homology to S2P is described by Rawson et al., supra and is listed in GenBank (GI1559384). Additionally, GenBank has listed a protein predicted from the C elegans genome as having HMG-CoA reductase homology (GI3875380).
The use of invertebrate model organism genetics can greatly facilitate the elucidation of biochemical pathways, and the identification of molecules that can modulate such pathways. Accordingly, it is an object of the invention to provide invertebrate nucleic acids and polypeptides involved in the SREBP pathway. It is also an object of the invention to provide invertebrate model organisms, including novel mutant phenotypes, for the study of lipid metabolism in general, and more particularly, for the elucidation of the SREBP pathway. It is a further object of the invention to provide methods for screening molecules that modulate lipid metabolism and/or the function of genes and proteins involved in the SREBP pathway.
These and other objects are provided by flies and nematodes that have been genetically modified to express or misexpress an SREBP pathway gene, for example using transposon mutagenesis, RNA interference, chemical mutagenesis, or other genetic techniques, In certain embodiments, expression of the SREBP pathway protein is driven by a heterologous promoter that is tissue-specific, developmentally-specific, or inducible, so that the effects of the expression or mis-expression can be observed in specific tissues, at certain developmental stages, or at specified times, respectively. Additionally, the SREBP pathway protein may be linked to one or more selectable markers that allows detection of expression. Typically, the expression of the SREBP pathway protein results in an identifiable phenotype. In the case of nematodes, the invention provides novel methods for the in vivo measurement of lipid content using BODIPY-fatty acid conjugates. The animal models can be used in genetic screens to identify other genes involved in lipid metabolism. They can also be used for screening small molecule libraries directly on whole organisms for possible therapeutic or pesticide use.
The invention also provides novel isolated nucleic acids (SEQ ID NOs:1, 3, and 5). and the SREBP pathway proteins encoded thereby (SEQ ID NOs:2, 4, and 6, respectively), as well as derivatives and fragments thereof. Methods are provided for constructing vectors containing the isolated nucleic acids. Such vectors can be used for making the animal models of the invention. They can also be introduced into host cells to be used for a variety of purposes including two-hybrid screening assays, production of SREBP pathway proteins, screening small molecules that affect lipid synthesis or metabolism, etc.